PROJECT SUMMARY Malaria remains an important global cause of morbidity and mortality, with 216 million cases and 445,000 deaths in 2016. Most of the malaria deaths are due to infection by the Plasmodium falciparum parasite. Resistance to all current anti-malarial medications is an emerging problem. The long-term goals of my research are to discover novel targets for new anti-malarials and to understand the function of these essential targets in the malaria parasite life cycle. The parasite genome is fully sequenced, but the functions for more than half of the parasite genes remain unknown. These genes are not similar to human genes (or other well-studied organisms). We hypothesize that this set of ?unknown genes? includes many genes that 1) are essential for parasite replication and 2) could be targeted by novel anti-malarials. Thus, there is an unmet public health need to discover a prioritized list of essential genes from this large number of ?unknown genes?. We have developed a robust pipeline to discover essential genes using an unbiased method to query the entire parasite genome. The goal of the current R21 exploratory/developmental application is to test the feasibility of this screening method. We can culture parasites with robotic liquid handling devices, allowing high-throughput sterile culture of ~20,000 individual (and genetically different) P. falciparum clones at the same time. We have created a strain of P. falciparum that allows quantitative monitoring of parasite replication in our high-throughput system. These two innovations allow us to perform genome-wide functional studies. In this application, we propose to identify parasites with temperature-sensitive mutations in essential genes. We will randomly mutate parasites with a chemical agent. Individual parasite clones will be cultivated in our high-throughput system at the permissive temperature and then tested for lack of growth at the restrictive temperature. Mutations will be identified by whole-genome sequencing, re-introduced into wild type parasites, and the novel essential genes will be functionally characterized. In Aim 1, we will evaluate the first 17 clones already isolated in our pilot screen. In the second aim, we will iteratively conduct the screen to identify and characterize 25-50 additional essential genes. This prioritized set of genes will be the starting point to develop a range of new anti-malarials.